Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56966—Animal cells
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
  • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
  • B01F33/30—Micromixers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/34—Purifying; Cleaning
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54386—Analytical elements
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
  • G01N33/548—Carbohydrates, e.g. dextran
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
  • G01N33/553—Metal or metal coated
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
  • B01F25/00—Flow mixers; Mixers for falling materials, e.g. solid particles
  • B01F25/40—Static mixers
  • B01F25/42—Static mixers in which the mixing is affected by moving the components jointly in changing directions, e.g. in tubes provided with baffles or obstructions
  • B01F25/43—Mixing tubes, e.g. wherein the material is moved in a radial or partly reversed direction
  • B01F25/431—Straight mixing tubes with baffles or obstructions that do not cause substantial pressure drop; Baffles therefor
  • B01F25/4317—Profiled elements, e.g. profiled blades, bars, pillars, columns or chevrons
  • B01F25/43172—Profiles, pillars, chevrons, i.e. long elements having a polygonal cross-section
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
  • B01F25/00—Flow mixers; Mixers for falling materials, e.g. solid particles
  • B01F25/40—Static mixers
  • B01F25/42—Static mixers in which the mixing is affected by moving the components jointly in changing directions, e.g. in tubes provided with baffles or obstructions
  • B01F25/43—Mixing tubes, e.g. wherein the material is moved in a radial or partly reversed direction
  • B01F25/431—Straight mixing tubes with baffles or obstructions that do not cause substantial pressure drop; Baffles therefor
  • B01F25/43197—Straight mixing tubes with baffles or obstructions that do not cause substantial pressure drop; Baffles therefor characterised by the mounting of the baffles or obstructions
  • B01F25/431971—Mounted on the wall
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0636—Focussing flows, e.g. to laminate flows
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
  • B01L2200/0652—Sorting or classification of particles or molecules
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/069—Absorbents; Gels to retain a fluid
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
  • B01L2400/0478—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules

Definitions

• the invention is generally directed to medicine and engineering. More specifically, the field is directed to isolation of biological materials, such as cells, for tissue engineering and regenerative medicine.
• Cellular isolation techniques are an essential component in studying specific populations, allowing for growth, genomic, and proteomic investigations.
• the detachment of cells adhered to any surface requires the application of a force that is greater in magnitude to that of adhesion.
• Fluid shear forces have been shown to be a simple method for cell detachment. Although this is a local and simple method of cell release, excessive exposure to fluid shear results in cell damage and reduction in viability.
• An alternative approach is to cleave the protein ligand that is bound to the capture surface using enzymes, such as trypsin.
• enzymes such as trypsin
• enzymatic exposure can cause morphological changes due to a disruption of the cell membrane and glycocalyx, leading to losses in cellular activity.
• enzymatic digestion has been shown to directly affect both the behavior and chemical makeup of the cells themselves.
• FACS fluorescent activated cell sorting
• MCS magnetic activated cell sorting
• the present disclosure relates to compositions and methods for the capture and release of biological materials, such as cells.
• the capture is highly specific.
• the disclosed hydrogel compositions comprise a plurality of alginic acid molecules and a plurality of branched polymer molecules.
• the plurality of alginic acid molecules is conjugated to or blended with the branched polymer molecule or one or more binding agents to form a hydrogel, and each of the branched polymer molecules comprises a plurality of groups.
• at least one group of each branched polymer molecule is conjugated to an alginic acid molecule and at least one other group of each branched polymer molecule is conjugated to one or more binding agents.
• the branched polymer molecule is a polyethylene glycol molecule. In some embodiments, the polyethylene glycol molecule is a four-arm molecule.
• the one or more binding agents is an antibody, antibody fragment, peptidomimetic compound, peptide, small molecule, or nucleic acid.
• the antibody is selected from the group consisting of antibodies against GPR49, LGR5, CD24, FLK1, CD45, CD31, CD34, and sca-1.
• the disclosed methods capturing and releasing target biological materials from a sample comprise providing a microfluidic device comprising one or more chambers for receiving fluids, wherein at least one of the one or more chambers comprises a surface coated with a hydrogel composition.
• the hydrogel composition comprises a plurality of alginic acid molecules and a plurality of branched polymer molecules in which the plurality of alginic acid molecules is conjugated to or blended with the branched polymer molecule or one or more binding agents to form a hydrogel.
• each of the branched polymer molecule comprises a plurality of groups, at least one group of each branched polymer molecule is conjugated to an alginic acid molecule and at least one other group of each branched polymer molecule is conjugated to one or more binding agents.
• the methods further comprise introducing a sample comprising target and non-target biological materials into the one or more chambers under conditions effective to bind the target biological materials to the hydrogel composition and releasing the target biological materials using a releasing agent.
• the methods further comprise removing the unbound non-target materials from the sample.
• the disclosed methods of capturing and releasing target biological materials from a sample comprise providing a microfluidic device comprising one or more chambers for receiving fluids, wherein at least one of the one or more chambers comprises at least one surface coated with a hydrogel composition.
• the hydrogel composition comprises a plurality of alginic acid molecules and a plurality of branched polymer molecules in which the plurality of alginic acid molecules is conjugated to the branched polymer molecule or one or more binding agents to form a hydrogel.
• each of the branched polymer molecule comprises a plurality of groups, at least one group of each branched polymer molecule is conjugated to an alginic acid molecule and at least one other group of each branched polymer molecule is conjugated to one or more binding agents.
• the methods comprise introducing a sample comprising target biological materials into a first chamber of the device under conditions effective to bind biological materials to the hydrogel composition and releasing the bound biological materials using a releasing agent.
• the methods also comprise contacting the releasing agent with a neutralizing agent to neutralize the releasing agent in a second chamber and providing the contents of the second chamber into a third chamber comprising a surface coated with the hydrogel composition, wherein the binding agent in the third chamber is a different binding agent than that used in (a), under conditions effective to bind the target biological materials to the hydrogel composition.
• the methods comprise releasing the bound, target biological materials using a releasing agent.
• the methods further comprise adding culture medium to the released biological materials.
• the methods further comprise repeating (d) through (f) using a different binding agent.
• the methods further comprise detecting the target biological materials after release from the hydrogel composition.
• the biological materials used in the disclosed methods are cells, proteins, solutes, or particulates, and wherein the releasing agent is a chelating agent, an enzyme, or a combination thereof.
• the cells are adult stem cells, fetal stem cells, progenitor cells, peripheral hematopoietic stem cells, endothelial progenitor cells, circulating tumor cell, mature circulating endothelial cells, amniotic stem cells, mesenchymal stem cells, adipose-derived stem cells, intestinal stem cells, skin stem cells, neural stem cells, or cancer stem cells.
• the cell is a living cell captured from the sample.
• the chelating agent used in the disclose methods is selected from the group consisting of EDTA, EGTA, and sodium citrate.
• the disclosed methods further comprise maintaining the living cell under conditions effective to culture, detect, analyze, or transform the living cell.
• methods of making a hydrogel composition comprise reacting branched polymer molecules with one or more binding agents in a buffer and reacting the branched polymer -binding agent solution with at least one alginic acid molecule to form a functionalized hydrogel.
• the functionalized hydrogel comprises each of the branched polymer molecules conjugated to one or more binding agents and further conjugated to at least one alginic acid molecule.
• the branched polymer molecule is a polyethylene glycol molecule. In some embodiments, the polyethylene glycol molecule is a four-arm molecule.
• the one or more binding agents is an antibody, antibody fragment, peptidomimetic compound, peptide, small molecule, or a nucleic acid.
• the antibody is selected from the group consisting of antibodies against GPR49, LGR5, CD24, FLK1, CD45, CD31, CD34, and sca-1.
• the disclosed microfluidic device comprises a substrate and one or more chambers for receiving a sample comprising target biological materials.
• the one or more chambers comprise a surface coated with a hydrogel composition, the hydrogel composition comprising a plurality of alginic acid molecules and a plurality of branched polymer molecules.
• the plurality of alginic acid molecules is conjugated to or blended with the branched polymer molecule or one or more binding agents to form a hydrogel.
• each of the branched polymer molecule comprises a plurality of groups, at least one group of each branched polymer molecule is conjugated to an alginic acid molecule.
• each branched polymer molecule is conjugated to a binding agent.
• a mixing chamber is also included for mixing bound target biological materials with a neutralizing agent.
• one or more additional surfaces are coated with a hydrogel composition.
• the hydrogel composition comprises a plurality of alginic acid molecules and a plurality of branched polymer molecules in which the plurality of alginic acid molecules is conjugated to the branched polymer molecule or one or more binding agents to form a hydrogel.
• each of the branched polymer molecule comprises a plurality of groups, at least one group of each branched polymer molecule is conjugated to an alginic acid molecule.
• at least one other group of each branched polymer molecule is conjugated to a binding agent that is different from the binding agent in step (i).
• the branched polymer in the disclosed devices is polyethylene glycol molecule.
• the polyethylene glycol molecule is a four-arm molecule.
• the one or more binding agents used in the disclosed devices is an antibody, antibody fragment, peptidomimetic compound, peptide, small molecule, or nucleic acid.
• the antibody is selected from the group consisting of antibodies against GPR49, LGR5, CD24, FLK1, CD45, CD31, CD34, and sca-1.
• Figure 1 is a diagrammatic representation of an infrared spectra of PEG- and antibody-functionalized hydrogels (Gels II-VII) compared to a standard solution of antibody (0.1 mg/ml and 0.05 mg/ml antibody). Note that the measurement is a bulk measurement.
• Figure 2 is a diagrammatic representation of a qualitative measurement of accessible antibody within hydrogel-coated micro fluidic devices.
• FIG. 3A is a diagrammatic representation showing the yield of endothelial progenitor cells (EPCs) captured from whole blood within microfluidic devices coated with PEG- and antibody-functionalized hydrogels.
• EPCs endothelial progenitor cells
• Figure 3B is a diagrammatic representation showing the purity of EPCs captured from whole blood within microfluidic devices coated with PEG- and antibody-functionalized hydrogels.
• Figures 4A-C are graphic representations showing structural differences in different gel types.
• all reagents including PEG, antibody, alginic acid
• Gel Type V utilizes a two-step protocol in which the PEG, antibody, EDC, and sulfo-NHS are combined in a single first step.
• Gel Types VI -VII has pre-mixing of PEG and antibody prior to mixing other components.
• Figures 5A-C depict qualitative representations of injected and released suspension pre- and post- micro fluidics array. Injected population (depicted in Figure 5 A) was constrained to a concentration of 100,000-200,000 cells/ml due to settling effects within the chip (depicted in Figure 5B) at respective concentrations. In Figure 5C, cells were released into 24-well plates, and a noticeable decrease in cellular density was observed. Scale bar represents ⁇ .
• Figures 6A-D illustrate that optimization of antibody-functionalized alginate allowed for improved capture efficiency and purity yields.
• Figure 6 A shows that the samples and formulations were divided into five scenarios, each varying one variable.
• Figure 6B compares the purity yield of these scenarios against the injected population. Quantifying the percent purity was preformed via flow cytometry against the injected ( Figure 6C) and the released ( Figure 6D) cells.
• Figures 7A-F shows age progression of released cells against unenriched population in the absence of Wnt3a protein. Unenriched organoid progression (depicted by Figures 7A-C) yielded significant larger cyst-like organoids surrounded by extraneous populations.
• Figures 7D depicts the expansion of single cell at day 2
• Figure 7E depicts induced hyperplasia at day 3
• Figure 7F depicts small lumen formation noticed with surrounding secreted apoptotic cells, at day 4.
• Figures 8A-D depict enriched and unenriched organoid in the presence of Lgr5 basal media constituents and Wnt3a.
• Figure 8 A shows that the unenriched population did not have an any increase in plate efficiency in the presence of Wnt3a.
• Figure 8C shows that the majority of organoids formed in the injected culture expressed a cyst- like structure harboring apoptotic cells.
• Figure 8B shows that the enriched population did have an increase in plating efficiency leading to more single derived organoids proliferating.
• Figure 8D shows that enriched cells exhibited similar morphology to the wnt3a absence study (d) at analogous time points.
• Figures 9A-D shows confocal compressed images illustrating the enriched organoid after the disclosed microfluidic isolation technique was used.
• Figure 9C depicts lumen formed indicative of the hollow nature in the spherical organoid.
• Figure 9A depicts apical localization of CD24, which indicates significant Sox9 expression exhibiting quiescence.
• Figure 9B depicts that the isolation capture antibody, GPR49/Lgr5 (b), was prevalent within the central domain, but expression was lower in comparison to CD24.
• Figures 10A-D represent unenriched organoid confocal images compressed in the z- plane. Organoid was extracted from matrigel after 4 days in culture.
• Apopotic cells are notable within the central lumen (Figure IOC), while the morphology of the organoid is spherical and planar.
• CD24 expression ( Figure 10A) is apical localized in the central domain and in varying levels of intensity. GPCR49/Lgr5 is present at lower intensity in locations where CD24 is expressed, arrows indicating . The notable presence of GPCR49/Lgr5 expression was trumped by CD24 overlay ( Figure 10D).
• Figure 11 A-D represent the sequence of devices for one embodiment of the adhesion-based micro fluidic separation of cells against multiple surface markers. Following capture and release from the device ( Figure 11 A), cells expressing marker 1 enter a device ( Figure 1 IB) where a calcium chloride solution is co-injected to neutralize the ethylene diamine tetraacetic acid (EDTA) present in the cell suspension. Another portion of the device ( Figure 11C) mixes the calcium chloride solution and cell suspension. Finally, in Figure 1 ID, the chamber captures cells against marker 2, which can then be eluted out using an injection of EDTA solution.
• Figure 1 IB a calcium chloride solution is co-injected to neutralize the ethylene diamine tetraacetic acid (EDTA) present in the cell suspension.
• EDTA ethylene diamine tetraacetic acid
• Figure 12 represents the performance of the multistage capture-release device system in dual-marker separation.
• the present disclosure relates to compositions and methods for highly specific capture and release of biological materials, such as cells.
• Hydrogel compositions comprising a plurality of alginic acid molecules conjugated to or blended with branched polymer molecules or one or more binding agents to form a hydrogel are disclosed.
• the disclosed methods and compositions provide surface coatings for the selective capture of a target cell type from a heterogeneous suspension with the additional capability to release captured cells nondestructively.
• the formulations and techniques disclosed herein allow for altered chemical compositions of alginate hydrogels, which have the ability to bind and release cells but which are prone to significant non-specific cell adhesion, with branched polymers such as poly(ethylene glycol) (PEG) or branched polymers with chemical functional groups known to suppress cell and protein adhesion, including but not limited to fluorocarbons and silicones.
• the incorporation of the branched polymer into the hydrogel structure is carried out in a way that also enables the functionalization of the alginate pre-polymer with a binding agent (e.g., an antibody, antibody fragment, peptidomimetic compound, peptide, small molecule, or nucleic acid) to provide specificity of capture.
• a binding agent e.g., an antibody, antibody fragment, peptidomimetic compound, peptide, small molecule, or nucleic acid
• the synthesis technique is designed for in situ assembly of the hydrogel within confined structures, such as microfluidic channels.
• the assembly techniques disclosed herein enables coating of channels made from any material, without a requirement for a particular type of material.
• Flow cytometric analyses of cells captured and detached using this approach from whole blood have indicated that the process is chemically and biologically nondestructive; specifically, there is no or little change in cell viability or phenotypic identity.
• the inclusion of the branched polymer, such as PEG within the hydrogel structure overcomes many of the problems associated with known hydrogel capture systems.
• the literature describes the design of surface coatings that can facilitate cell detachment when an external stimulus is applied, such as an electrical potential or a small temperature change.
• An example of the former is a surface coating that consists of ligands bound to the surface via an electroactive chemical functional group.
• the electroactive quinoine ester undergoes a chemical change to lactone upon applying an electrical potential.
• This approach requires electrode incorporation into the capture device and careful optimization of release parameters.
• a thermally-responsive polymer such as poly(N- isopropylacrylamide), which is hydrophobic at 37°C and hydrophilic at 20 °C, is another recently-described approach.
• the hydrophobic surface is adhesive to cells and its
• the disclosed methods and devices allow release of target cells from substrates either in static cell culture or flow-based cell separation.
• the instant disclosure does not require mechanical, enzymatic, electrical or optical interfaces for cell detachment.
• the disclosed methods and devices can be used without extensive physical or chemical perturbations to the biological environment. Prior techniques, on the other hand, require an external stimulus or require physical or chemical perturbations that compromise, for example, the cellular environment.
• the instant disclosure can be used to selectively capture and release biological materials to isolate, for example, stem and progenitor cell populations. The isolated populations can be used for seeding on engineered scaffolds. Engineered replacement organs, and regenerative medicine generally, require pure populations of rare cells to produce a functional organ.
• compositions of the disclosure can be alternately formulated to comprise, consist of, or consist essentially of, any appropriate components disclosed in this disclosure.
• the compositions of the disclosure can additionally, or alternatively, be formulated so as to be devoid, or substantially free, of any components, materials, ingredients, adjuvants or species used in the prior art compositions or that are otherwise not necessary to the achievement of the function and/or objectives of the present disclosure.
• a "hydrogel” is a three-dimensional, semi-solid network of one or more molecules in which a relatively large amount of water is present.
• the hydrogel can be a polymer.
• a "polymer” is a structure composed of monomers.
• “Monomers” are molecules having one or more groups that can react with each other or other types of monomers to form a polymer.
• a non-limiting example of a monomer is vinyl chloride, which can give a plastic known as "vinyl.”
• Another non-limiting example of a vinyl monomer is acrylamide which can give a gel known as a polyacrylamide gel.
• compositions comprising alginate hydrogels in which alginic acid is in the presence of divalent cations. Such compositions are capable of easily dissolving in the presence of chelators.
• the presently disclosed hydrogels are biocompatible and can be functionalized (i.e., conjugated) with cell-adhesive molecules.
• the alginate hydrogels can be functionalized with binding agents.
• binding agent means a molecule that binds to another molecule or complex structure. Binding agents include antibodies, antibody fragments, peptidomimetic compounds, peptides, small molecules, and nucleic acids. Antibodies are selected from the group consisting of antibodies against GPR49, LGR5, CD24, FLK1, CD45, CD31, CD34, sca-1, and various other proteins.
• the alginate hydrogels can also include branched polymers, such as polyethylene glycol ("PEG") or branched polymers with chemical functional groups known to suppress cell and protein adhesion, including but not limited to fluorocarbons and silicones.
• PEG polyethylene glycol
• the PEG can conjugated to or blended with (that is, functionalized) binding agents.
• the PEG can be conjugated to or blended with alginic acid molecules to form a hydrogel.
• the hydrogels utilize 4-arm PEG molecules with primary amine terminations at the end of each arm.
• a 4-arm PEG molecule has four attachment points for functionalization with other agents such as alginic acid, binding agents, or linkers.
• each 4-arm PEG molecule binds to a carboxylic acid group to the alginate hydrogel backbone, leaving up to three primary amine groups for functionalization with a binding agent.
• the 4-arm arrangement allows for triple the binding agent (e.g., antibody) content of the hydrogel and provides protection against non-specific cell binding relative to non PEG-y-lated alginate hydrogels.
• Methods of making hydrogel compositions comprise reacting polyethylene glycol molecules with one or more binding agents in a buffer and reacting the polyethylene glycol-binding agent solution with at least one alginic acid molecule to form a functionalized hydrogel, the functionalized hydrogel comprising each of the polyethylene glycol molecules conjugated to one or more binding agents and further conjugated to at least one alginic acid molecule.
• the methods described herein ensure that at least one attachment point in a branched polymer, such as a PEG molecule, is available for binding with an alginate gel matrix, leaving at least another attachment point for functionalization with binding agents, such as antibodies.
• binding agents include but are not limited to antibodies against GPR49, LGR5, CD24, FLKl, CD45, CD31, CD34, sca-1, and various other proteins.
• the methods involve conjugating alginic acid to a binding agent such as an antibody and providing the
• antibody/alginic acid conjugate to a branched polymer such as PEG to forma hydrogel.
• the alginic acid-antibody conjugate is reacted with amine-terminated PEG molecules.
• the amine-terminated PEG molecule is a 4-arm PEG molecule.
• the binding agent, antibody, and PEG are reacted at the same time to create an antibody/alginic acid/PEG hydrogel.
• the PEG and binding agent are conjugated. In these embodiments, the conjugate is reacted with alginic acid.
• the methods further comprise utilizing protecting groups, such as fluorenylmethyloxycarbonyl (FMOC) groups, to achieve control over binding agent conjugation to primary amine groups.
• protecting groups such as fluorenylmethyloxycarbonyl (FMOC) groups
• the methods also comprise adding the antibody/alginic acid/PEG hydrogels to a micro fluidic device to coat the inner surface of the device.
• the hydrogel is allowed to form in situ and coats the inner surfaces of one or more chambers of the device.
• microfluidic devices comprising a substrate; and one or more chambers for receiving a sample comprising target biological materials, the one or more chambers comprise a surface coated with a hydrogel composition, the hydrogel composition comprising a plurality of alginic acid molecules and a plurality of polyethylene glycol molecules in which each of the polyethylene glycol molecule comprises a plurality of groups, at least one group of each polyethylene glycol molecule is conjugated to an alginic acid molecule and at least one other group of each polyethylene glycol molecule is conjugated to a binding agent.
• the devices disclosed herein further comprise a mixing chamber for mixing bound target biological materials with a neutralizing agent and one or more additional surfaces coated with a hydrogel composition.
• the hydrogel composition comprises a plurality of alginic acid molecules and a plurality of polyethylene glycol molecules in which each of the polyethylene glycol molecule comprises a plurality of groups and at least one group of each polyethylene glycol molecule is conjugated to an alginic acid molecule.
• At least one other group of each polyethylene glycol molecule is conjugated to a binding agent that is different from the binding agent in step (i).
• the substrate is a silica-containing material (e.g., glass, PDMS).
• the substrate is a polymeric material (both biocompatible and non-biocompatible), and the polymer is either bonded to itself or to other silica substrates.
• the substrate is a thermosetting plastic, such as epoxies, including fiber-reinforced plastics.
• the substrate is a metal (for example, gold, silver, platinum, copper, aluminum); metal alloy; metal oxide (copper oxide, aluminum oxide, silver oxide, indium tin oxide, etc.); an inorganic material, including but not limited to semiconductors and magnetic materials.
• the substrate is a combination of the silica, polymeric, metallic, or inorganic materials described herein.
• Microfluidic devices known in the art can also be utilized for the methods disclosed herein.
• the methods can be used to separate, for example, EPCs from blood for subsequent use in vascular tissue engineering or cell-based regenerative repair of vascular tissue in vivo.
• the methods involve allowing an alginic acid/PEG hydrogel to form in situ in a microfluidic device.
• the methods further entail providing a sample to the device and allowing the binding agent conjugated to the hydrogel to capture a target biological material, such as a particular cell type. The sample is allowed to pass through the device and the captured cells are released using a releasing agent.
• the releasing agent is a release buffer including, for example, a chelator such as ethylenediammetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), and sodium citrate.
• EDTA ethylenediammetetraacetic acid
• EGTA ethylene glycol tetraacetic acid
• samples include but are not limited to whole blood, serum, saliva, lymph, bile, urine, and any other biological fluid.
• methods of capturing and releasing target biological materials from a sample comprise providing a microfluidic device comprising one or more chambers for receiving fluids, wherein at least one of the one or more chambers comprises a surface coated with a hydrogel composition.
• the hydrogel composition comprises a plurality of alginic acid molecules and a plurality of branched polymer molecules in which each of the branched polymer molecule comprises a plurality of groups and at least one group of each branched polymer molecule is conjugated to an alginic acid molecule. Furthermore, at least one other group of each branched polymer molecule is conjugated to one or more binding agents.
• the methods further comprise introducing a sample comprising target and non-target biological materials into the one or more chambers under conditions effective to bind the target biological materials to the hydrogel composition and releasing the target biological materials using a releasing agent.
• the methods further comprise removing the unbound non-target materials from the sample.
• methods of capturing and releasing target biological materials from a sample comprise (a) providing a microfluidic device comprising one or more chambers for receiving fluids, wherein at least one of the one or more chambers comprises at least one surface coated with a hydrogel composition.
• the hydrogel composition comprise a plurality of alginic acid molecules and a plurality of branched polymer molecules, wherein each of the branched polymer molecule comprises a plurality of groups, at least one group of each branched polymer molecule is conjugated to an alginic acid molecule, and at least one other group of each branched polymer molecule is conjugated to one or more binding agents.
• the methods comprise (b) introducing a sample comprising target biological materials into a first chamber of the device under conditions effective to bind biological materials to the hydrogel composition; (c) releasing the bound biological materials using a releasing agent; and (d) contacting the releasing agent with a neutralizing agent to neutralize the releasing agent in a second chamber.
• the methods entail (e) providing the contents of the second chamber into a third chamber comprising a surface coated with the hydrogel composition, wherein the binding agent in the third chamber is a different binding agent than that used in (a), under conditions effective to bind the target biological materials to the hydrogel composition; and (f) releasing the bound, target biological materials using a releasing agent.
• Figures 11 A-D illustrate the devices and methods using multiple chambers.
• a sample was injected via a syringe pump into the first alginate-based capture stage ("Marker 1 isolation"/ Figure 11 A).
• This stage was connected to stage B, which was a 2- way valve. In its "closed” configuration, this valve allowed the waste from stage A to pass through to a collection tube. After the waste went through, the waste stream was closed using, for example, a pinch valve.
• Figure 1 IB The purpose of the calcium chloride was to neutralize the EDTA in the cell suspension emerging from stage ( Figure 11 A).
• the combined output (which was in laminar flow) was sent into a mixing chamber (Figure 11C) containing herringbone features.
• the mixed solution then entered stage ( Figure 1 ID), where the cells expressing receptors for the second capture molecule were captured.
• the final step in the separation process was the injection of an EDTA solution into the stage A ( Figure 11 A) inlet, which releases the captured cells from stage B ( Figure 1 IB). This solution was collected in a tube containing an excess of culture medium to minimize any deleterious effect of the EDTA on the cells.
• the methods further comprise adding culture medium to the released biological materials. In some embodiments, (d) through (f) can be repeated using a different binding agent. In some embodiments, the methods further comprise detecting the target biological materials after release from the hydrogel composition. In some embodiments, the methods further comprise maintaining the cells under conditions effective to culture, detect, analyze, or transform the cells, including living cells.
• the cells are rare cells, including but not limited to adult stem cells, fetal stem cells, progenitor cells, peripheral hematopoietic stem cells, endothelial progenitor cells, circulating tumor cell, mature circulating endothelial cells, amniotic stem cells, mesenchymal stem cells, adipose-derived stem cells, intestinal stem cells, skin stem cells, neural stem cells, and cancer stem cells.
• the cell is a living cell captured from the sample.
• the chelating agent is selected from the group consisting of EDTA, EGTA, and sodium citrate.
• Figures 4A-C illustrates various synthetic methods for the production of hydrogels (designated gel types I through VII). The progressive improvement in EPC capture yield and purity from gel type II-VII is shown.
• all reagents including PEG, antibody, alginic acid
• Gel Type V utilizes a two- step protocol in which the PEG, antibody, EDC, and sulfo-NHS are combined in a single first step.
• Gel Types VI- VII has pre-mixing of PEG and antibody prior to mixing other components. Pre-mixing allows optimal dispersion of antibody molecules among the PEG chains.
• Figure 3A-B depict results after 300 of whole blood collected in heparin tubes was directly injected into individual micro fluidic devices, and 10 devices were run in parallel. Cells released from each device were pooled into a single suspension to allow enumeration by flow cytometry. Data reported represent yield and purity for EPCs recovered from a total blood volume of 3 mL. Error bars denote standard deviations based on 3 independent measurements of EPC and total cell counts made with the same sample. Increased yield and purity were observed with the incorporation of 10k MW PEG (gel types II vs. IV).
• the methods and compositions disclosed can also be used with 20k MW PEG, as well as other molecular weight PEG molecules so long as size constraints, such as steric forces, do not affect cell binding efficiency.
• the first step in the synthesis was the combination of PEG and antibody with the coupling agents EDC and sulfo-NHS prior to the addition of alginic acid in the second step.
• gel type V provides slightly greater EPC capture but with a lower degree of scatter, indicating better mixing of the antibody molecules with the PEG.
• the accessible antibody content of gel type V is similar to that of gel type IV ( Figure 2),
• a bicinchoninic acid (BCA) assay kit was utilized to measure the relative amount of antibody accessible to a solution flowing through each device. A lower absorbance is associated with a greater amount of accessible antibody. Error bars denote standard errors based on 8 independent measurements for each gel type. Better mixing also allows for more effective interspersing of PEG and antibody molecules on the hydrogel surface, which is consistent with the higher EPC purity obtained with gel type V relative to gel type IV. Fewer PEG particles were observed in the PEG- and antibody- functionalized alginic acid solution, which is consistent with better PEG- antibody mixing.
• the two-step synthesis protocol for gel types VI and VII allows for pre-mixing by providing time for antibody and PEG molecules to mix 'undisturbed' without the constraining presence of EDC and sulfo-NHS.
• pre-mixing is not necessarily required for the methods and compositions disclosed herein, longer mixing time can improve EPC capture performance in terms of yield and purity, as can be seen when comparing gel types VI and VII to gel type V.
• the longer mixing and incubation times provided for gel type VII relative to gel type VI provided the good yield ( ⁇ 10 4 EPCs recovered) and purity (74%) as well.
• Example 1 describes methods and compositions for the highly specific capture and release of biological materials, such as cells.
• the capture antibody, monoclonal mouse anti- human CD34, and goat anti-human FLK-1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
• Anti-human CD133-PE, anti-human CD45-FITC, and anti-goat IgG-PerCP antibodies were obtained from eBioscience (San Diego, CA).
• Rabbit IgG was purchased from Vector Labs (Burlingame, CA).
• Calcium chloride dihydrate and alginic acid were purchased from Sigma (St. Louis, MO).
• Amine-terminated 4-arm PEG (PEG-NH2) with molecular weights of 10,000 (10k MW) and 20,000 (20k MW) were purchased from Laysan Bio (Arab, AL).
• the device used a post array design similar to that used by Nagrath et al, Nature, 450 (7173), 123-U10 (2007).
• the posts were arranged in a hexagonal layout as described by Gleghorn et al, Lap Chip, 10(1), 27-29 (2010).
• the posts had a diameter of 100 ⁇ and a transverse spacing of 150 ⁇ from center to center. Rows had a center to center spacing of 125 ⁇ and each is offset by 50 ⁇ .
• the post array was 0.7 cm long and 0.5 cm wide.
• the posts heights were approximately 50 ⁇ for the devices fabricated by soft lithography as described below.
• PDMS poly(dimethyl siloxane)
• the silicone elastomer and curing agents were mixed in a 10: 1 (w/w) ratio and poured on top of the negative master wafers, degassed, and allowed to cure overnight at 65°C.
• PDMS replicas were then pulled off the wafers prior to punching inlet and outlet holes with a 19-gauge blunt-nose needle.
• the replicas and glass slides were exposed to oxygen plasma (100 mW with 8% oxygen for 30 s) in a PX-250 plasma chamber (March Instruments, Concord, MA) and immediately placed in contact with each other.
• the irreversible bonding between PDMS and glass was completed by baking for 5 min at 65°C.
• Gel Types I-VII Seven different hydrogel formulations were investigated in this study, and these are designated as Gel Types I-VII.
• Gel Type I 45 mg of alginic acid, 4.8 mg EDC, 13.2 mg sulfo-NHS, and 20 inert IgG (1 g/mL) were added to 2 ml of MES buffer solution and mixed using an IKA Ultra Turrax Tube Disperser for 29 min and allowed to incubate for 60 min.
• Gel Type II 45 mg of alginic acid, 4.8 mg EDC, 13.2 mg sulfo-NHS and 100 anti-human CD34 (200 ⁇ g/mL) were added to 2 mL of MES buffer, mixed as before, and incubated for 60 min.
• Gel Type III 45 mg alginic acid, 4.8 mg EDC, 13.2 mg sulfo-NHS, 22.5 mg 20k MW PEG, and 100 ⁇ ⁇ anti sheep CD34 were added to 2 mL of MES buffer, mixed for 29 min, and allowed to incubate for 60 min.
• Gel type IV consisted of 45 mg alginic acid, 4.8 mg EDC, 13.2 mg sulfo-NHS, 22.5 mg 10k MW PEG, and 100 ⁇ anti sheep CD34 added to 2 mL of MES buffer, mixed for 29 min and allowed to incubate for 60 min.
• Gel Type V was created by mixing 4.8 mg EDC, 13.2 mg sulfo-NHS, 22.5 mg 10k MW PEG, and 100 ⁇ , anti sheep CD34 in 2 ml of MES buffer for 29 min and then adding 45 mg of alginic acid followed by 29 min of mixing and 60 min of incubation.
• Gels VI and VII were formed by mixing 22.5 mg 10k MW PEG with 100 ⁇ ⁇ antibody in 2 mL of MES buffer and mixing for 10 min and 29 min, respectively, and incubating for an additional 15 min and 60 min, respectively.
• 4.8 mg EDC, 13.2 mg sulfo-NHS, and 45 mg alginic acid were then added to the mixture, mixed for 29 min and allowed to incubate for 60 min.
• each functionalized alginic acid solution for each gel type was injected into a Slide- A-Lyzer Dialysis Cassette 10,000 molecular weight cut-off (Fisher) and dialyzed against MES buffer for 48 hours to remove unreacted sulfo-NHS and EDC.
• Table 1 summarizes the synthetic steps and components for each gel type. Steps 1 and 2 indicate the sequential nature of the protocol followed for combining the respective reagents. Table 1. Summary of Synthesis Protocols for Different Hydrogel Formulations.
• a 1 g/mL solution of CaCl 2 in deionized water was injected into each device (by hand, using a 1 mL syringe) and allowed to incubate overnight.
• the CaCl 2 solution was then withdrawn by hand using a 1 mL syringe.
• the PEG- and antibody-functionalized alginate solution prepared for each gel type was then injected into the devices by hand and allowed to adsorb for 1 hour.
• the devices were rinsed with MES buffer at ⁇ /min for 10 min using a Harvard Apparatus PHD 2000 syringe pump (Holliston, MA), followed by a lOOmM CaCl 2 solution in MES buffer at 10 ⁇ /min for 10 min to form a thin layer of hydrogel on the walls of the microchannels. Finally, the devices were rinsed with MES buffer at 5 ⁇ /min for 10 min to remove unreacted CaCl 2 .
• a BCA protein assay solution was prepared according to manufacturer instructions. The solution was then injected into each device at 5 ⁇ /min for 40 min. The output was collected in a microplate and absorption at 562 nm was measured using a Bio-Tek Powerwave XS spectrometer.
• EPC enumeration cells released from each device were mixed with 10 ⁇ each of anti-human CD 133 PE, anti-human CD45 FITC, anti-goat FLK-1, and anti-goat IgG PerCP. The mixture was stored in the dark for 30 min and centrifuged at 130 x g for 10 min. The supernatant was decanted and cells were suspended in 200 ⁇ , of PBS for enumeration using a Beckman Coulter Cell Lab Quanta SC flow cytometer. Cells that were CD133+, CD45-, and FLK-1 + were counted as EPCs. Results
• Figure 1 shows infrared spectroscopy data for quantification of antibody loading within the functionalized alginic acid solutions emerging from the one- or two-step synthesis protocol.
• all of the alginic acid solutions have comparable antibody content between 0.05 and 0.06 mg/mL.
• Figure 2 shows the relative total protein measurements made using a BCA assay kit.
• the BCA solution becomes more transparent as it comes in contact with proteins such as antibodies.
• proteins such as antibodies.
• the protein content of the solutions exiting the devices is shown as a function of gel type in Figure 2 and is expressed in arbitrary units of absorbance rather than as a calibrated mass or concentration.
• the relative measurement allows comparison of the accessible anti-CD34 capture antibody between each gel type.
• Figure 2 shows an increase in accessible antibody from gel types I- VII while the total amount of antibody added to the mixture remains constant ( Figure 1), indicating an increase in the efficiency of conjugation between the gelled surface and the antibody.
• Figure 3 shows yield and purity data for the capture of EPCs from whole blood using the hydrogel-coated microfluidic devices.
• gel type I which has an inert antibody conjugated to it, shows negligible EPC adhesion as expected.
• Gel type II which contains the anti-CD34 antibody, shows significantly higher EPC adhesion relative to gel type I (p ⁇ 0.005), albeit with a high degree of scatter.
• the purity of capture achieved with gel type II is, however, relatively low (-23%; Figure 3B).
• the effect of adding the 4-arm PEG to the hydrogel structure is shown clearly by comparing gel types II and IV, whose synthesis protocol is otherwise identical.
• gel types V-VII a two step protocol for combining reagents was followed.
• the conjugation of the antibody molecules to the 4-arm PEG is carried out first before introducing alginic acid.
• This formulation improved yield and purity of EPC capture relative to gel type IV.
• the two-step protocol was modified such that EDC and sulfo-NHS were added in the second step with alginic acid and the first step was restricted to the mixing together of PEG and antibody.
• short times were provided for mixing and incubation for the first step (10 min and 15 min, respectively, for gel type VI), the yield did not improve relative to gel type V, but purity was higher.
• Example 2 discloses devices and methods for the microfluidic capture and release of intestinal stem cells using two binding agents, specifically, GPR49 and Lgr5 antibody receptors.
• this Example allows for multiplexing and larger sample volume to be processed while retaining viability of the eluded target population.
• culture methods have been developed to induce hyperplasia and organoid forming units derived from single cells.
• Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 2009, 459 (7244), 262-U147). These cells do not require a mesenchymal niche to develop into these units and rely on growth factors to induce differentiation cues.
• Intestinal tissue samples were obtained from neonatal Lewis rats. Large intestine was extracted, split laterally, and fragmented into 1 mm segments. Fragmented tissue was incubated in 2 mM EDTA at 4°C for 30 minutes. Tissue samples were separated from the solution and placed in 20 mL of phosphate buffered saline (PBS, Gibco) for 10 minutes of agitation. The supernatant fluid was then collected and centrifuged at 150xg for three minutes; the pellet was collected, suspended in 10 mL of serum-free Dulbecco's Modified Eagle's Medium (DMEM, Cellgrow) and centrifuged again at 150xg. The pellet was suspended in 5 mL of serum- free DMEM solution and filtered through a 100 ⁇ cell strainer. The solution was then filtered through 20 ⁇ cell strainers into 1 mL eppendorf tubes.
• PBS phosphate buffered saline
• Microfluidic devices were fabricated using traditional soft lithography at the George J. Kostas Nanoscale Technology and Manufacturing Research Center at Northeastern
• Antibody- functionalized alginate reaction underwent six different scenarios but stoichiometric ratios of reagents remained constant through out each scheme.
• MES pH was altered for each respective scenario which was either held at ph 4.7 or 6.0; the pH was titrated with NaOH to a pH of 6.0. The amalgam was allowed to incubate for 60 minutes in scenario II, but the subsequent reagents were added immediately in the remaining scenarios.
• Micro fluidic devices with a hexagonal post array were utilized for cell separation. Each device was filled with alginate functionalized with Anti-GPCR GPR49 and allowed to incubate for 60 minutes. Channels were formed by flowing through 100 of pH 6 MES buffer at 10 ⁇ / ⁇ , 100 of 100 mM CaCl 2 at 10 ⁇ / ⁇ , and 100 ⁇ ⁇ of 0.1% bovine serum albumin at 10 ⁇ / ⁇ . A Harvard Apparatus syringe pump was used to obtained precise flow rates. Cell solutions obtained were mixed to ensure homogenous suspension and 200 ⁇ , were drawn into 1 mL syringes.
• Lgr5 basal media contained the following constituents: Advanced DMEM F-12, 5 ml N2 supplement, 10 mL B27 without vit. A, 5mL HEPES, 6.25 mL glutamax. Each sample was rinsed with 350 ⁇ ⁇ of Lgr5 basal media in to remove EDTA from the cell culture. Then ⁇ ⁇ ⁇ of ROCK inhibitor (y-27632, Sigma-Aldrich) was added to 10 mL of Lgr5 media.
• Enriched organoids were fixed with 4% paraformadahyde and rinsed with 2mM glycine in PBS. 6 U/ml dispase (stem cell technologies) was added and incubated for 1 hour to release organoids from matrigel. Organoids were pipetted into 200 ⁇ ⁇ Lgr5 media blocking solution containing: 3% BSA, 10% goat serum, .1% triton X-100, lOmM HEPES, and lOmM glycine. 1 :50 of respective antibodies, anti-GPCR GPR49 and anti-CD24, to blocking solution was added and incubated at 4°C overnight.
• Organoids were pipetted out of solution and into 200 ⁇ of blocking solution containing normalized concentrations of Alexfluor 488, Alexafluor 568, and ⁇ g/ml DAPI for 3 hours. Organoids were mounted on glass cover slides and confocal images were taken via Nikon confocal microscope.
• Lgr5 positive cells Released enriched Lgr5 positive cells were imbedded in Matrigel and grown under similar conditions as described in Sato et al; Single Lgr5 stem cells build crypt- villus structures in vitro without a mesenchymal niche. Nature 2009, 459 (7244), 262-U147.
• the culture technique for lgr5 positive cells included growth factor constituents that were altered slightly to take into account species dependent factors. Rat endothelial growth factor (EGF) and murine rspondin-1 were used, in contrast to the literature sources that have implemented a hybridized mouse model. Y-26743, rock inhibitor, was used to improve culture stability and to prevent anoikis in a single cell suspension.
• EGF endothelial growth factor
• Y-26743 rock inhibitor
• the inhibitor was also used concurrently in the microfluidic enrichment technique, and it was observed to result in an increase in plating efficiency (data not shown), but exhibited little affect in unenriched cultures (Figure 7A-C). Progression of organoids, from enriched single lgr5 cells, was viewed up to 4 days and compared against an unenriched population (Figure 7D-E). Growth was noticed at day 2 and progressed into hyperplasia stage at day 3. Small lumen formation coupled with an increase of hyperplasia is observed at day 4.
• Figure 7A-C show that unenriched organoid progression yielded significant larger cyst-like organoids surrounded by extraneous populations.
• Figures 7D-F show four-day progression of enriched organoid derived from single cell suspension. Expansion of single cell (Figure 7D) at day 2, induced hyperplasia at day 3 ( Figure 7E), and small lumen formation noticed with surrounding secreted apoptotic cells, at day 4 ( Figure 7F) are shown. Scale bars represent ⁇
• Enriched and unenriched organoids were released from culture at day 4 via a dispase treatment to degrade the Matrigel. Stained organoids were exposed to anti-GPR GPRCR49, anti- CD24, and DAPI, each conjugated with alexa fluor 488 (green) and 524 (red) ( Figures 9 and 10). Confocal microscopy facilitated determination of the morphology of the organoids and protein expression. Unenriched organoids ( Figure 10) had a significant population of apoptotic cells within the central domain. The organoid did not undergo hyperplastia for the culture duration and exhibited a bright CD-24 signal in an elliptical pattern.
• Anti-Lgr5/GPRCR49 expression was faint ( Figure 10b), and expression was limited to the lumendomain of the organoid. Localization of anti-Lgr5/GPRCR49 diminished in the significantly low CD24 populations( Figure 10A-B).
• the topography of the unenriched culture exhibited an elliptical planar morphology (Figure 10D) in contrast to the enriched organoid, which was spherical ( Figure 9D).
• the central domain expressed CD-24(green) and anti-Lgr5/GPCR49 (red), localized in the apical membrane ( Figure 9A-B).
• CD-24 expression was localized along 4 different membranes ( Figure 9A), and expression was lower in intensity compared to the unenriched organoid.
• Localized anti- Lgr5/GPCR49 were centered in the apical membrane and expressed in 2 membranes ( Figure 9B). Expression of both markers was strictly limited to the central domain, coinciding with Sox9 (CD-24) and Lgr5 genomic trends.
• the instant disclosure fulfills the need in developing a cost-effective and fluorescent- free cell isolation devices and methods for application such as tissue engineering.
• Conventional methods in intestinal stem cell isolation rely on hybridized mice models and complex
• the instant Example describes a micro fluidics method that enriches intestinal stem cell populations using alginate coupled with anti-GPCR49/Lgr5.
• the enriched lgr5 cells have been grown in appropriate culture medium. After adding the cells in medium, CD24 expression coinciding with Lgr5 expression in the organoid central domain was investigated.
• This Example describes methods and devices that enrich a select target population while retaining viability, expression, and growth morphology.
• This Example describes a one-pass microfluidic alginate capture and release model capable of a 24-fold enrichment to a GPCR49/Lgr5 purity of 49%.
• a pacifying agent BSA
• BSA a pacifying agent
• the phenomena generates a cascading affect in which coagulated cell types containing lgr5 positive cells adhere to the alginate coating; immediate injection of strained cells was performed to facilitate in dispersion.
• Chemical interactions and stability between alginate, EDC, 4-arm star PEG, Anti-GPCR49/Lgr5 were increased as the reaction pH became more basic.
• the disclosed methods allowed for fluorescent-label free isolation of intestinal stem cells while retaining similar growth morphology in situ.
• the unenriched population contain doublets of paneth-lgr5 postive cells, which sustain the necessary Wnt signaling; thus, a null effect was noticed in the presence of the cofactor.
• Wnt3a it was noted that the plating efficiency amongst the unenriched population was slightly higher than the enriched suspension; this being indicative of paneth cell niche signaling allowing for improved long-term organoid viability.
• Enriched organoids were plated in similar fashion to the injected suspension, but the significant difference resided in the morphological changes and plating efficiency of the released GPCR49/lgr5 positive cells.
• the images indicate a smaller organoid with a small central lumen formed yet to harbor any apoptotic cells.
• CD24 and GPCR49/Lgr5 expression was bound in the central domain with similar expression patterns as the latter.
• the presented images eluded to that the microfluidics enrichment process retained similar morphological outcomes as previously reported.
• the Example discloses methods and devices that can be used for cell sorting and tissue engineering.
• the Example describes an intestinal stem cell isolation technique from wild-type intestinal digestate.
• the current convention is limited to transgenic mice models and complex instrumentation to isolate these cells.
• the disclosed methods allow the end-user to isolate cell subtypes in a speedy process while retaining cell viability.
• This Example relates to compositions and methods for a multistage, highly specific capture and release of biological materials, such as cells.
• Figure 11 A-D represent a configuration of alginate-hydrogel based devices that include capture stages for each of two antibodies. In some embodiments, more than two antibodies are contemplated.
• FIG. 11 A-D a sample was injected via a syringe pump into the first alginate - based capture stage ("Marker 1 isolation'V Figure 11 A). This stage was connected to stage B, which was a 2-way valve. In its "closed” configuration, this valve allowed the waste from stage A to pass through to a collection tube. After the waste went through, the waste stream was closed using, for example, a pinch valve. ( Figure 1 IB). The purpose of the calcium chloride was to neutralize the EDTA in the cell suspension emerging from stage ( Figure 11 A). To ensure mixing of the calcium chloride solution with this cell suspension, the combined output (which was in laminar flow) was sent into a mixing chamber ( Figure 11C) containing herringbone features.
• the mixed solution then entered stage ( Figure 1 ID), where the cells expressing receptors for the second capture molecule were captured.
• the final step in the separation process was the injection of an EDTA solution into the stage A ( Figure 11 A) inlet, which releases the captured cells from stage B ( Figure 1 IB). This solution was collected in a tube containing an excess of culture medium to minimize any deleterious effect of the EDTA on the cells.
• This Example showed the ability of this dual-stage capture system to isolate endothelial progenitor cells (EPCs) from untreated whole blood.
• the objective was to capture cells that are CD34+/FLK1+.
• Figure 12 shows cell counts (obtained by flow cytometry) of the cells emerging from stage A and stage B.
• the various populations shown represent categories of CD34+ cells and the "total" column represents the total number of cells released.
• the objective of the second capture device was to remove CD34+ cells that do not express the second marker, FLK-1, namely the CD34+ cells that are also CD45+. The sharp decrease in the number of CD45+ cells coming out of the second capture stage relative to the first capture stage shows this enrichment.

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